ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEIC ACID.IV. LINKAGE OF SINGLE DEOXYNUCLEOTIDES TO THE DEOXYNUCLEOSIDE ENDS OF DEOXYRIBONUCLEIC ACID By Jutivs ApLER,* I. R. Lenman, Maurice J. Bessman,t E. S. Simms, AND — ARTHUR KoRNBERG DEPARTMENT OF MICROBIOLOGY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, ST. LOUIS, MISSOURI Communicated May 26, 1958 Although the synthesis of DNA! by an enzyme purified from Escherichia colt requires four deoxynucleoside triphosphates (dAPPP, dCPPP, dGPPP, TPPP) in addition to Mg++ and DNA,?: ? nevertheless a single deoxynucleoside triphosphate can be incorporated into DNA to an extent about one-thousandth of that for the complete reaction.? The number of the single deoxynucleotides incorporated is of the same order as an approximation of the number of DNA chains added to the reaction mixture. It was the purpose of this investigation to determine whether the single deoxy- nucleotide, incorporated in the absence of the other three deoxynucleotide substrates, was covalently linked to the DNA and, if so, where in the DNA chain. It was also of interest to establish whether only one or several deoxynucleotides were added to a DNA chain. The results show that the single deoxynucleoside tri- phosphate reacts with the deoxynucleoside end of a DNA chain to form a typical 3’,5’-phosphodiester linkage and that only one or a few nucleotides are added to a chain. METHODS AND MATERIALS Materials.— Uniformly labeled C'4-5’-deoxycytidylate was obtained by enzymatic hydrolysis of DNA from a Chromatium species grown on CQ, as the sole carbon source. Deoxyribonucleoside triphosphates were prepared as described previously.” Micrococcal DNase‘ was generously supplied by Dr. L. Cunningham. Bull semen 5’-nucleotidase® and purified human semen phosphomonoesterase were kindly provided by Dr. L. A. Heppel. Calf spleen phosphodiesterase was prepared ac- cording to Hilmoe and Heppel.6 Venom phosphodiesterase was prepared from the venom of Crotalus adamanteus according to Koerner and Sinsheimer’ and was free of phosphomonoesterase. The DNA-synthesizing enzyme used in these studies (Fraction VII) was purified about 2,000-fold.2 Calf thymus DNA was prepared according to Kay, Simmons, and Dounce.’ Preparation of DNA products.—Four products were prepared from dAP*PP, dCP#PP, TP#PP, or C!‘dCPPP, essentially as described previously,” except that the concentration of DNA was raised 25-fold in order to obtain greater amounts of incorporation. The reaction mixtures contained (per ml.) 6.0 mymoles of a deoxynucleoside triphosphate; 6.7 umoles of magnesium chloride; 67 umoles of potassium phosphate buffer at pH 7.5; 800 ug. of calf thymus DNA; and 20 ug. of Fraction VII enzyme. The volume of these reaction mixtures ranged from 18 to 36 ml. Incubations were carried out at 37° for 2.5 hours (for dAP#PP, dCP®PP, and TP#*PP) or for 1 hour (for C'4dCPPP). 641 642 BIOCHEMISTRY: ADLER ET AL. Proc. N. A. 8. The reactions were stopped and the products freed of unreacted deoxynucleoside triphosphate in one of two ways: (1) acidification with an equal volume of cold 1 N perchloric acid, then alternate solubilization of the precipitated product in 0.1 NV NaOH and precipitation in 0.5 N perchloric acid (when enzyme had been omitted, np radioactivity could be detected in this repeatedly precipitated product); (2) addition of sodium chloride to a final concentration of 0.2 M and heating at 70° for 10 minutes,’ then repeated dialysis against 0.2 M sodium chloride. The radio- activity in this dialyzed product was then entirely precipitable by perchloric acid. RESULTS Extent and Kinetics of Reaction —In the four DNA products prepared, the in- corporation of dAP®?, dCP#, TP#?, and C4dCP was 0.25, 0.13, 0.32, and 0.34 myumoles, respectively, per milliliter of reaction mixture. For an assumed molecular weight of 5 & 106 for thymus DNA, these amounts correspond to an incorporation of 0.5 to 1 mole of deoxynucleotide per mole of DNA. Several factors add un- certainty to this approximation: the polydispersity of DNA, lack of information about the size and number of competent DNA molecules, and the hydrolysis of DNA to smaller molecules during the incubation. TABLE 1* ULTRACENTRIFUGAL ANALYSIS OF PRODUCT Fraction Remaining Sedimentation Expt. Basis for in Top Compartment Coefficient oO. Calculation (Per Cent) (8) Schlieren boundary .. 12.3 Ll... Optical density at 260 mu 48 12.8 Radioactivity 48 13.2 Schlieren boundary .. 12.7 2.....00. Optical density at 260 my 50 12.7 Radioactivity 52 12.5 3f....... Optical density at 260 mz 10 22.1 Radioactivity 13 21.4 * In these experiments the product used was from a reaction with TP#2PP as the only deoxynucleoside tri- phosphate. The sedimentation coefficients were measured at a relatively high concentration (optical density at 260 my = 7.0) in order to achieve significant measurements of radioactivity. When the same product was run at a tenfold dilution, the sedimentation coefficient was 21 S. t In experiment 3 the product had been heated at 100° for 10 minutes. Asa result of the heating, the schlieren boundaries were too diffuse to serve as a basis for calculation of a sedimentation coefficient. A study of the time course of the reaction between DNA and a single deoxy- nucleoside triphosphate showed an initial rapid rate of incorporation followed by a slower rate. In a typical experiment with dCP#*PP as the only deoxynucleoside triphosphate, the incorporation into DNA at 30, 60, and 120 minutes was 4.0, 5.0, and 7.0 uumoles, respectively. This slow rate of incorporation after the initial rapid reaction could be explained by the production of new, reactive DNA ends. Such degradation of the DNA, catalyzed by a contaminating nuclease, is indicated by a small decrease in viscosity during the preparation of the products. Another contaminating nuclease has the property ascribed to snake venom phosphodiesterase (see below) of removing deoxynucleotides from the deoxynucleoside ends of DNA chains. Ultracentrifugal Analysis of the Product Showing the Incorporation of Deoxy- nucleotides into Molecules the Size of DNA.—In order to determine whether the incorporated radioactive deoxynucleotide possesses the same sedimentation rate as VoL. 44, 1958 BIOCHEMISTRY: ADLER ET AL, 643 the average DNA molecule, a product was examined in a fixed partition separation cell’ in the ultracentrifuge. Centrifugation was stopped after about half of the DNA had sedimented out of the top compartment of the separation cell, as determined by the schlieren optical system. Then the fractions of the optical density and radio- activity remaining in the upper compartment of the cell were measured, and sedi- mentation coefficients were calculated from these data and from the schlieren boundaries. As shown in Table 1, the sedimentation coefficients determined from the three measurements agree. Even after the products had been heated at 100° for 10 minutes, to try to break down the structure of the macromolecules,!! the radio- activity still sedimented at the same rate as the DNA. These data suggest that the radioactive deoxynucleotide had formed covalent linkages, rather than much weaker secondary bonds, to molecules of the same average size as DNA. Hydrolysis of Products to 3’-Deoxynucleotides Demonstrating 3’,5'-Phosphodi- ester Linkages between the Incorporated Deoxynucleotides and Deoxynucleoside Ends of DNA Chains.—To determine whether the incorporated P*? deoxynucleotide is bound to DNA by a phosphodiester linkage, a method of hydrolysis was sought which would leave the P®? with the adjacent deoxynucleotide (Fig. 1). For RNA such a result has been accomplished by hydrolysis to 2’- and 3’-ribonucleotides with alkali.!?:!3 For ~~] DNA no such reagent was available until the re- cent discovery and purification of a DNase from (P) Micrococcus pyogenes‘ and a phosphodiesterase from calf spleen.6 The successive action of these two enzymes has now resulted in nearly complete (P) hydrolysis of DNA to 3’-deoxynucleotides. Koer- ner and Sinsheimer have obtained a similar result by combining spleen DNase and spleen phospho- (P) diesterase.’ OOD In the present experiments the progress of hy- x drolysis was followed by measuring phosphate re- x) leased by human semen phosphomonoesterase. p-p--- Pp” — Micrococcal DNase in 0.01 M CaCl, and 0.1 M tris(hydroxymethyl)aminomethane at pH 8.5 hy- Cc drolyzed the products to acid-soluble fragments containing 55-65 per cent of their total phosphate Fic. 1.—Hydrolysis of product to in the monoester form. Spleen phosphodiesterase p ueoay nucleotides by the com- . : : ined action of micrococcal DNase then added to the digest (adjusted to pH 7.0 with nd spleen phosphodiesterase. P = HCl) converted 86-94 per cent of the total phos- pi preen posP , phate to the monoester level. No inorganic phos- phate (<2 per cent of the total phosphate) could be detected at the end of the di- gestion. The resulting 3’-mononucleotides, separated by chromatography, were sensitive to semen phosphomonoesterase but were entirely resistant to 5’-nucleo- tidase under conditions where 5’-deoxynucleotides were completely hydrolyzed. When products prepared from dCP#PP, dAP#PP, or TP*?PP as the only deoxynucleoside triphosphate were hydrolyzed in this manner, each of the four 3’-deoxynucleotides of thymus DNA was found to be radioactive (experiments 644 BIOCHEMISTRY: ADLER ET AL. Proc. N. A.§. 1-3, Table 2). This result shows that the single deoxynucleoside triphosphates had formed 3’,5’-phosphodiester linkages (Fig. 1) to each of the four deoxynucleo- tides of thymus DNA. When a product prepared from C'“dCPPP as the only deoxynucleoside tri- phosphate was hydrolyzed to 3’-deoxynucleotides, two-thirds of the recovered radioactivity appeared in deoxycytidine and one-third in 3’-deoxycytidylate (experiment 4, Table 2). Since deoxynucleosides formed in this hydrolysis could arise only from terminal deoxynucleotides of DNA (Fig. 1), two-thirds of the C'dCPPP must have been located at the deoxynucleoside ends of DNA chains. The remaining one-third probably had also reacted with ends of chains but was TABLE 2 Hyprotysis or Propucts To 3’-DEOXYNUCLEOTIDES ° Substrate for Per Cent of Expt. Preparation Products of Recovered No.* of Product Hydrolysis Radioactivity t 3’-Deoxyadenylate 18 ‘-Deoxycytidylate 34 1 aCP#PP 3’-Deoxyguanylate 23 ‘-Thymidylate 25 ‘-Deoxyadeny late 57 3’-Deoxycytidylate 6 2 dAP#2PP 3’-Deoxygunaylate 22 '-Thymidylate 15 3’-Deoxyadeny late 17 ’-Deoxycytidylate 10 3 TP#PpPp 3’-Deoxyguanylate 21 ’-Thymidylate 52 4 C“%dCPPP Deoxycytidine 67 3’-Deoxycytidylate 33 *In Experiments 1, 2, 3, and 4, respectively, specific activities at the time of chromatog- raphy were 5.4 X 107,7.5 X 107, 5.4 X 106, and 1.4 X 107c.p.m. per umole. — n Experiment 3 the hydrolyaate was desalted by adsorption on Norit and eluted with ammoniacal ethanol, and then the products of hydrolysia were separated by paper electro- horesis (Markham and Smith, Biochem. J., 52, 552, 1952). In Experimenta 1, 2, and 4 the hydrolysate was chromatographed on Dowex 1 formate columns (Cunningham, Catlin, and De Garilhe, J. Am. Chem. Soc., 78, 4642, 1956; and Sinsheimer, J. Biol. Chem., 208, 445, 1954), Deoxycytidine was determined as counts not held by a Dowex 1 formate or a Dowex 1 chloride column at pH 10. This radioactivity not held by the column contained less than 0.5 per cent of the total optical density of the 3’-deoxycytidylate and therefore could not represent 3’-deoxycytidylate unadsorbed due to overloading of the column. On the basis of the optical density at 260 my, the total amounts of 3’-deoxyadenylate, 3’- deoxycytidylate, 3/-deoxy guanylate, and 3’-thymidylate recovered in experiment 1 were 30.6, 22.1, 22.9, and 30.6 amoles, respectively. This represents 96 per cent of the optical density at 260 mp added to the column. The recoveries of radioactivity after chromatography or electrophoresis were 83, 93, 78, and 83 per cent in experiments 1, 2, 3, and 4, reapectively. followed by another C14-deoxycytidylate to produce terminal “runs” of C14- deoxycytidylate. This possibility is supported by the disproportionately high radioactivity found in 3’-P*-deoxycytidylate in experiment 1 of Table 2. Also in products prepared from dAP®*PP or TP®PP, ‘runs’ of the incorporated deoxy- nucleotide are indicated similarly by experiments 2 and 3 of Table 2. It cannot be decided from these data whether these “runs” occur frequently and are very short or whether they occur rarely and are long. Kinetics of Hydrolysis of Products to 5'-Deoxynucleotides Demonstrating the Location of Incorporated Deoxynucleotides at the Deoxynucleoside Ends of DNA Chains.—To confirm that the incorporated radioactive deoxynucleotides are located at the deoxynucleoside ends of DNA chains, a reagent was sought which would hydrolyze the products stepwise from the deoxynucleoside ends so as to Vou, 44, 1958 BIOCHEMISTRY: ADLER ET AL. 645 release all the radioactivity at a time when few of the total deoxynucleotides had been released. Laskowski et al.’ and Boman" have suggested that snake venom phosphodiesterase attacks DNA stepwise from one end. Recently, Khorana et al.* have shown that this phosphodiesterase hydrolyzes synthetic thymidine oli- gonucleotides stepwise from the deoxynucleoside end, and with thymidine oligo- nucleotides bearing a 3’-acetyl group at the deoxynucleoside end the first product to appear was the 3’-acety] derivative of 5’-thymidylate. Singer, Hilmoe, and Heppel” have obtained a similar result with oligoribonucleotides. When the products were treated with venom phosphodiesterase, nearly all (>90 per cent) of the radioactiv- ity was liberated as acid-soluble deoxynucleotides at a time when less than 3 per cent of the de- oxynucleotides had been released, judged by the appearance of acid- soluble ultraviolet absorbing ma- terial (Fig. 2). Such a result was obtained for products prepared from dAP#PP, TP#PP, or C“dCPPP. This finding pro- vides independent evidence that the radioactivity in these prod- ucts is located at or near the de- oxynucleoside ends of DNA 0 chains. It may be inferred from 100-4 cn Radioactivity “J nn l 25-7 Per Cent of Total Optical Density ——S 60 2 180 these kinetic studies that the phosphodiesterase does not re- main attached to a DNA mole- cule, degrading it sequentially to completion, but that, instead, other DNA molecules compete favorably with the polydeoxy- nucleotide product of the enzyme action. DISCUSSION In these experiments a reaction Time in Minutes Fie. 2.—Action of venom phosphodiesterase on product. The product used in this experiment was ‘om a reaction with C'dCPPP as the only deoxy- nucleoside triphosphate. The reaction mixture con- tained 3 mg. of product, 2 units’ of snake venom phos- phodiesterase, 50 ymoles of MgCl, and 200 «moles of tris(hydroxymethy])aminomethane at pH 8.5 in 5.0 ml. At the times indicated, 0.5-ml. aliquots were removed, 0.25 mg. of DNA was added as carrier, and the reac- tions were stopped by the addition of 0.5 ml. of 1 N cold perchloric acid. Radioactivity and optical density at 260 my were measured in the supernatant fractions. At 200 minutes, 50 more units of diesterase were added, which resulted in total release of both radioactivity and optical density in leas than 3.5 hours. has been observed in which a DNA molecule (containing about 20,000 deoxy- nucleotides) adds at its deoxynucleoside end one or a very few molecules of a single deoxynucleoside triphosphate to form a 3’,5'-phosphodiester bond (Fig. 1). Several alternative mechanisms appear to have been eliminated. 1. A non-covalent binding of the deoxynucleoside triphosphate to the DNA is excluded by the demonstrated phosphodiester linkage between the incorporated deoxynucleotide and the three other deoxynucleotides of DNA. 2. Pyrophosphorolysis of the added DNA to supply the missing three deoxy- nucleoside triphosphates, followed by de novo synthesis of DNA chains, seemed from the start an unlikely hypothesis for explaining the reaction between DNA and 646 BIOCHEMISTRY: ADLER ET AL. Proc. N. A.S. a single deoxynucleoside triphosphate, because levels of inorganic pyrophosphate 100 times greater than those formed in the reaction are necessary to produce significant pyrophosphorolysis.? This possibility was eliminated by the finding that the incorporated deoxynucleotide is located exclusively at or near the ends of DNA chains. 3. De novo formation of chains containing exclusively the single deoxynucleo- tide is eliminated by the demonstrated phosphodiester linkage between the single deoxynucleoside triphosphate and each of the three other deoxynucleotides of thymus DNA. 4. Reaction of the single P**-deoxynucleoside triphosphate with the non- nucleoside end of the DNA chain" would not have resulted in the appearance of P*? in the three other deoxynucleotides of DNA when the product was hydrolyzed to 3’-deoxynucleotides. The basis of a sensitive tool for analyzing the number and kinds of end-groups of a homogeneous DNA could be furnished by reacting such a DNA sample with a single deoxynucleoside triphosphate and then hydrolyzing the product to 3’- deoxynucleotides. Since nucleases which contaminate the purified enzyme could produce new ends or remove deoxynucleotides added in the reaction, extension of these studies in this direction must await further purification of the enzyme. What is the pertinence of this reaction between DNA and a single deoxynucleo- side triphosphate to the extensive DNA synthesis observed when all four deoxy- nucleoside triphosphates are present? It appears likely that it is the same highly purified EZ, colt enzyme which is responsible for both reactions. One possibility is that the reaction between DNA and a single deoxynucleoside triphosphate may actually be the first step in the over-all process of enzymatic replication of the DNA primer; then the terminal replicating chain, formed when four deoxynucleoside triphosphates are present, would loop back, in order to orient itself along the primer chain. It is conceivable that such loops could be produced by the rotation of a very few deoxynucleotide units and could be dissipated at a later time by nuclease action. Another possibility is that the reaction between DNA and a single deoxy- nucleoside triphosphate is merely part of the extension of the shorter of two un- equally long chains of a DNA double helix and is not directly related to the replica- tion of intact DNA molecules. In this event replication of DNA would not involve a covalent binding between the primer and the replicating chain; instead, the deoxynucleoside triphosphates would first form hydrogen bonds to corresponding deoxynucleotides of the primer chain and would then polymerize. It is evident that more must be learned about the chemistry of the DNA primer itself as well as about these enzymatic reactions before these questions of mechanism can be answered. SUMMARY 1. The reaction between deoxyribonucleic acid (DNA) and a single radioactive deoxynucleoside triphosphate resulted in a product whose radioactivity sediments at the same rate as the average DNA molecules; this finding indicates that the deoxynucleotide had become incorporated into molecules which have the same size as DNA. 2. Hydrolysis of the product to 3’-deoxynucleotides demonstrated the formation VoL. 44, 1958 BIOCHEMISTRY: ADLER ET AL. 647 of 3’,5’-phosphodiester linkages between the deoxynucleoside end of a DNA chain and the added deoxynucleotide. These studies further showed that only one or a very few molecules of the single deoxynucleoside triphosphate reacted with the end of each chain. 3. The kinetics of hydrolysis of the product by snake venom phosphodiesterase, involving a sequential liberation of deoxynucleotides from the deoxynucleoside end of DNA chains, provided independent evidence that the deoxynucleoside triphos- phate reacted with the deoxynucleoside end of a DNA chain. This investigation was supported by research grants from the National Institutes of Health, Public Health Service, and the National Science Foundation. We are grateful to Dr. Eloise E. Clark for performing the ultracentrifugal analyses and to Dr. Howard K. Schachman for valuable advice and discussions. * Life Insurance Medical Research Fund Postdoctoral Fellow. t Postdoctoral Fellow of the National Cancer Institute. Present address: McCollum-Pratt Institute, The Johns Hopkins University, Baltimore, Maryland. 1 Abbreviations used: DNA, deoxyribonucleic acid; DNase, deoxyribonuclease; dAPPP, deoxyadenosine triphosphate; dCPPP, deoxycytidine triphosphate; dGPPP, deoxyguanosine triphosphate; TPPP, thymidine triphosphate. ? I. R. Lehman, M. J. Bessman, E. S. Simms, and A. Kornberg, J. Biol. Chem. (in press, vol, 233) *M. J. Bessman, I. R. Lehman, E. S. Simms, and A. Kornberg, J. Biol. Chem. (in press, vol. 233). “L. Cunningham, B. W. Catlin, and M. P. de Garilhe, J. Am. Chem. Soc., 78, 4642, 1956. 'L. A. Heppel and R. J. Hilmoe, Methods in Enzymology, 2, 547, 1955. 6 Tbid., p. 565. 7 J. F. Koerner and R. L. Sinsheimer, J. Biol. Chem., 228, 1049, 1957. A unit is that amount of enzyme which in 2 hours at 37° produces 1 »mole of monoesterified phosphate from a pancreatic DNase limit digest. *E. R. M. Kay, N.S. Simmons, and A. L. Dounce, J. Am. Chem. Soc. 74, 1724, 1952. 5 A. Tiselius, K. O. Pederson, and T. Svedberg, Nature, 140, 848, 1937. 0H. K. Schachman, Methods in Enzymology, 4, 32, 1957. 11C. A. Dekker and H. K. Schachman, these Proceepinas, 40, 894, 1954. 11 C, Heidelberger, E. Harbers, K..C, Leibman, Y. Takagi, and V. R. Potter, Biochim. et Biophys. Acta, 20, 445, 1956. SE. 8. Canellakis, Biochim. et Biophys. Acta, 25, 217, 1957. 14M. Laskowski, G. Hagerty, and U. Laurila, Nature, 180, 1181, 1957. 1H. G. Boman, Nature, 180, 1182, 1957. 1° H. G. Khorana, G. M. Tener, W. E. Razzell, and R. Markham, Fed. Proc. 17, 253, 1958. 17M. F. Singer, R. J. Hilmoe, and L. A. Heppel, Fed. Proc., 17, 312, 1958. 18 A. Kornberg, Advances in Enzymol., 18, 191, 1957. 19R. Markham and J. D. Smith, Biochem. J., 52, 552, 1952. 0 R. L. Sinsheimer, J. Biol. Chem., 208, 445, 1954.